The First Report of Postharvest Stem Rot of kohrabi Caused by Sclerotinia sclerotiorum in Korea

Mycobiology
Research Note
The First Report of Postharvest Stem Rot of
Kohlrabi Caused by Sclerotinia sclerotiorum in
Korea
Joon-Young Kim 1 , Md. Aktaruzzaman 1 , Tania Afroz 1 , Young-Il Hahm 2 and Byung-Sup Kim 1, *
Department of Plant Science, Gangneung-Wonju National University, Gangneung 210-702, Korea
Daegwallyong Horticultural Cooperative, Pyeongchang 232-950, Korea
1
2
Abstract In March 2014, a kohlrabi stem rot sample was collected from the cold storage room of Daegwallyong Horticultural
Cooperative, Korea. White and fuzzy mycelial growth was observed on the stem, symptomatic of stem rot disease. The pathogen
was isolated from the infected stem and cultured on potato dextrose agar for further fungal morphological observation and to
confirm its pathogenicity, according to Koch’s postulates. Morphological data, pathogenicity test results, and rDNA sequences of
internal transcribed spacer regions (ITS 1 and 4) showed that the postharvest stem rot of kohlrabi was caused by Sclerotinia
sclerotiorum. This is the first report of postharvest stem rot of kohlrabi in Korea.
Keywords
Kohlrabi, Pathogenicity, Postharvest disease, Sclerotinia sclerotiorum, Sclerotinia stem rot
Kohlrabi (Brassica oleracea var. gongylodes) is a low, stout
cultivar of the cabbage that will grow almost anywhere. It
develops a thickened stem just above the soil line. Although
this vegetable is often called a “turnip-rooted cabbage,” the
edible portion is an enlarged stem rather than root tissue.
Kohlrabi originated in northern Europe in the 16th century.
Sclerotinia sclerotiorum (Lib.) de Bary is among the
world’s most successful and omnivorous fungal pathogens,
affecting a wide range of vegetable and field crops in many
countries. Diseases caused by S. sclerotiorum occur in all
stages of plant growth, including in seedlings and mature
plants, and in post-harvest products [1, 2]. It has been
reported that the fungus attacks more than 400 different
species of plants [3, 4], and in Korea alone, more than 59
plant species have been reported as being hosts for S.
sclerotiorum [5]. Cool and humid conditions favor the
Mycobiology 2014 December, 42(4): 409-411
http://dx.doi.org/10.5941/MYCO.2014.42.4.409
pISSN 1229-8093 • eISSN 2092-9323
© The Korean Society of Mycology
*Corresponding author
E-mail: bskim@gwnu.ac.kr
Received September 10, 2014
Revised October 13, 2014
Accepted October 13, 2014
This is an Open Access article distributed under the terms of the
Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted
non-commercial use, distribution, and reproduction in any medium,
provided the original work is properly cited.
occurrence of Sclerotinia rot in the field, during transit and
in storage, and the optimal temperatures for fungal growth
range from 15 o C to 21 o C [6]. In Korea, this fungus has
been associated with Sclerotinia rot in various vegetable
crops in the field, but has not been reported as a postharvest
disease [7]. The objective of this study was to identify the
causal agent associated with postharvest Sclerotinia stem
rot disease in kohlrabi, based on culture characteristics,
molecular phylogenetics, and pathogenicity.
Isolation of fungi and pathogenicity test. In March
2014, symptoms of rot were observed on kohlrabi in the cold
storage room of Daegwallyong Horticultural Cooperative,
Pyeongchang, Gangwon Province, Korea (Fig. 1A). Specifically,
white and fuzzy mycelial growth was seen on the stems.
Infected stems were collected in sterilized plastic polythene
bags and transported to the laboratory for pathogen isolation.
They were then cut into small pieces, approximately 0.5 to
1 cm in size. The pieces were then surface-sterilized with
1% (v/v) sodium hypochlorite (NaOCl) for 1 min, washed
3 times with sterile distilled water, and then dried with
sterilized filter paper. Next, the pieces were placed in Petri
plates containing potato dextrose agar (PDA) medium
(Difco, Detroit, MI, USA) and incubated at 20 ± 2 o C for 5
days. For pure culture isolation, the mycelia grown on the
PDA plates were used to inoculate fresh PDA plates.
To determine the pathogenicity of the fungus, 1 mycelial
disc (7 days old, 6.5 mm) was placed into a small cut of
kohlrabi stem and put in to a plastic box lined with moist
filter papers. This was then incubated at 20 ± 2 o C, under
laboratory conditions. After 5 days, white mycelia were
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410 Kim et al.
morphological characteristics as those of the original isolates
(Fig. 1B). Thus, the fungal pathogen fulfilled the criteria
stipulated by Koch’s postulates and was identified as the
causal agent of postharvest Sclerotinia stem rot of kohlrabi.
Sclerotia germination and sexual structure. For
sclerotia germination, sandy loam soil was placed in a Petri
dish, and 30-day-old 10~15 sclerotia were washed, dried,
and pressed down to a depth of approximately 2 mm from
the top of the Petri dish. The Petri dish was then kept
shaded from sunlight, at 20 ± 2 o C, and watered 3 times a
week with sterilized water. The plate was exposed to natural
sunlight after the appearance of the first stipe initial [8, 9].
Fully developed apothecia were removed for further study.
The asci and ascospores were examined microscopically
and were photographed at 40× magnification.
Fig. 1. A, Diseased kohlrabi showing fuzzy growths of
mycelium on the stem; B, Kohlrabi inoculated with Sclerotinia
sclerotiorum developed Sclerotinia stem rot symptoms after 5
days of incubation; C, Three-week-old colony and black
sclerotia of S. sclerotiorum growing on potato dextrose agar
medium; D, Apothecia; E, Ascus containing 8 ascospores; F,
Ascospores (scale bars: E, F = 20 µm).
seen on the stem, along with symptoms of rot. The fungal
pathogen was re-isolated from the disease lesions on the
inoculated stem; this re-isolated pathogen exhibited the same
DNA extraction, polymerase chain reaction (PCR),
and sequence analysis. For DNA extraction, mycelia
were grown in 250-mL flasks containing 100mL of potato
dextrose broth, which were incubated for 6 days at 25 o C on
a rotary shaker at 135 rpm. Mycelia were harvested by
vacuum filtration through Whatman grade 1 filter paper
and then lyophilized for 24 hr before grinding them to a
fine powder. Next, 100 mg of the ground powder was
transferred to a 1.5-mL Eppendorf tube, and DNA was
extracted using the CTAB extraction method [10].
The extracted DNA was used for PCR sequencing of
rDNA genes by using universal primers for internal
transcribed spacer (ITS) 1 (5'-TCCGTAGGTGAACCTGCGG-
3') and ITS 4 (5'-TCCTCCGCTTATTGATATGC-3') [11].
The amplification was performed in a 25 µL reaction
mixture containing 0.5 µL of each primer, 0.5 µL of Taq
DNA polymerase (Bioneer, Daejeon, Korea), 0.5 µL of each
dNTP, 2.5 µL of 10× PCR reaction buffer, 18.5 µL of
distilled water, and 2.0 µL of template DNA. The reaction
was performed in an Eppendorf Mastercycler (Eppendorf,
Hamburg, Germany) under the following conditions: predenaturation
at 94 o C for 5 min, followed by 35 cycles of
denaturation at 94 o C for 35 sec, annealing at 52 o C for
Table 1. Morphological and culture characteristics of Sclerotinia sclerotiorum isolated from kohlrabi
Characteristics Study isolate S. sclerotiorum a
Colony Color White to gray White to gray
Sclerotium Shape Initially cushion-like or globularor irregular,
dark brown, finally black
Initially cushion-like or short-cylindrical
and white, finally black
Apothecium Shape Cup-shaped with yellowish brown color Cup-shaped with yellowish brown color
Size (cm) 0.8~1.4 0.5~2.0
Ascus Shape Cylindrical, hyaline, 8-spored Cylindrical, hyaline, 8-spored
Length (µm) 103.5~142.7 110~160.0
Width (µm) 5.7~7.7 6.0~10.0
Ascospore Shape Hyaline, ellipsoid to ovoid Hyaline, ellipsoid to ovoid
Length (µm) 8.9~11.2 9.0~14.0
Width (µm) 4.4~5.3 4.0~6.0
a
Described by Kohn [12].

Postharvest Stem Rot of Kohlrabi 411
Fig. 2. Neighbor-joining phylogenetic tree of Sclerotinia sclerotiorum strain SS3 and related species identified by the internal
transcribed spacer gene sequences from the GenBank database. Numbers at the nodes indicate bootstrap values from a test of
1,000 replicates. The scale bar indicates the number of nucleotide substitutions. Evolutionary analyses were conducted using the
MEGA5 program [13].
55 sec, and elongation at 72 o C for 1 min, followed by a final
extension at 72 o C for 10 min [14]. The obtained nucleotide
sequences were used in a BLASTN search of the GenBank
database (http://www.ncbi.nlm.nih.gov/BLAST/). Phylogenetic
analysis of S. sclerotiorum was performed using the MEGA5
program, with the neighbor-joining method [13]. Sequence
data were deposited in GenBank (accession No. KM656466).
Identification and characterization of S. sclerotiorum.
In total, 7 fungal strains were obtained from the kohlrabi
with Sclerotinia rot, and of these, strain SS3 was examined
for identification. Strain SS3 was identified as S. sclerotiorum
by analyzing the morphological characteristics of the isolated
fungus and performing rDNA sequencing analysis. The
fungus produced white to gray colonies on PDA medium
incubated at 20 ± 2 o C for 7 days, which after 3 wk showed
small sclerotia that were produced on the peripheries of the
plates (Fig. 1C). These sclerotia were black in color and
globose, cylindrical, or irregular in shape. Apothecia were
obtained from germinating sclerotia on sand medium after
5~6 wk incubation at 20 ± 2 o C (Fig. 1D). Eight elliptical
ascospores of uniform size were observed in each ascus
under the microscope (Fig. 1E and 1F), which is a typical
characteristic of S. sclerotiorum [15]. The morphological
characteristics of the identified species are summarized in
Table 1. The ITS sequences obtained in this study were
compared to GenBank database sequences by using the
NCBI BLAST search tool. The sequences identified based on
rRNA-ITS alignment were 100% similar to those of several S.
sclerotiorum species (accession Nos. KJ817041.1, KF148605.1,
JQ618848.1, and HM769811.1). Thus, S. sclerotiorum was
identified as the causative agent of postharvest Sclerotinia
stem rot of kohlrabi in this study (Fig. 2), which constitutes
the first report of this disease in kohlrabi in Korea.
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